Methods and compositions of insect control

ABSTRACT

The invention describes recombinant DNA sequences transcribed into RNA constructs capable of forming Virus Like Particles (VLPs) suitable for insect control applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. application Ser. No. 16/062,097, filed Jun. 13, 2018, which is a National Stage Entry of PCT Application No. PCT/US2016/065408, filed Dec. 7, 2016, which claims priority from U.S. Provisional Application No. 62/273,654, filed Dec. 31, 2015, the contents of all of which are incorporated herein by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The entire contents of a paper copy of the “Sequence Listing” and a computer readable form of the sequence listing entitled Insect Control Sequence Listing ST25.txt, which is 30 kilobytes in size and was created on Dec. 7, 2016, are herein incorporated by reference.

FIELD OF THE INVENTION

The invention comprises methods and compositions relating to virus-like particles (VLPs) containing heterologous cargo molecules capable of generating an RNAi-mediated gene suppression effect on targeted insects. Such compositions and methods have application in crop protection and other aspects of insect control.

BACKGROUND OF THE INVENTION

RNAi-mediated gene suppression, first described in the nematode C. elegans, has been shown to be an effective method for modulating gene expression in many other organisms. Fire et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806 (1998). The role of RNAi in controlling proliferation of insects affecting crops has been demonstrated using double-stranded RNA (dsRNA) by a number of research groups. Reviewed in, Ivashuta, et al. Environmental RNAi in herbivorous insects. RNA 21:840 (2015). Recombinant RNA constructs used for RNAi purposes described in the prior art generally consist of dsRNAs of about 18 to about 25 base pairs (siRNAs), but also include longer dsRNAs (long dsRNAs) usually between about 100 to about 1,000 base pairs (bp). To successfully introduce dsRNA into insects, dsRNAs longer than or equal to approximately 60 bp are required for efficient uptake when supplied in the insect's diet. Bolognesi, et al. Ultrastructural Changes Caused by Snf7 RNAi in Larval Enterocytes of Western Corn Rootworm (Diabrotica virgifera virgifera Le Conte) PLoS One 7:e47534 (2012). Long dsRNA molecules are cleaved in-vivo into a diverse population of siRNAs by the host's Dicer enzyme complex. Alternatively, RNAi gene suppression can also occur through the action of anti-sense RNAs directed to specific sequences via related processes. Practical application of RNAi methods for controlling insects in the field is limited by the cost of in vitro RNA synthesis and the chemical fragility of RNA, even dsRNAs, to environmental and enzymatic degradation.

Bacteriophage MS2 capsid mediated delivery of toxins and imaging agents to human cancer cells has been shown to be an effective method for delivering such agents to eukaryotic cells in vitro. Ashley, et al., Cell-specific delivery of diverse cargos by bacteriophage MS2 virus-like particles. ACS nano 5:5729 (2011). Whether such bacteriophage capsids can serve a similar function for delivery of RNAi precursors to insects in the field is unknown. Effective delivery of RNAi precursors into target insects requires preventing non-specific RNA degradation, a facile route of administration, and the ability to release the RNAi precursors at the appropriate point within the target insect such that the RNAi precursors can be taken up by the insect cells and properly processed. Ideally, the RNAi precursor and delivery system must be economical and relatively simple to produce and distribute. The invention described herein satisfies all these criteria and have the added benefit of allowing rapid discovery, prototyping and commercial-scale production of new RNAi molecules.

SUMMARY OF THE INVENTION

The invention described herein uses the unique properties of VLPs (alternatively known as APSE RNA Containers, or “ARCs”), to provide an improved system for delivering long dsRNA and RNAi precursors (dsRNAi) which can be processed intra-cellularly to produce siRNA for suppressing expression of a target gene, preferably in an insect host, more preferably a Coleopteran or Lepidopteran insect pest. Of particular interest are Coleoptera such as bark beetle, elm leaf beetle, Asian longhorn beetle, death watch beetle, mountain pine beetle, coconut hispine beetle, the various corn rootworms, and the Colorado potato beetle. RNAi methods of controlling Colorado potato beetle are especially desired since these beetles have developed resistance to virtually all known insecticides.

Coleopteran insect pests are known to be susceptible to RNAi introduced via the gut, either by direct injection or by feeding on plant matter treated with RNAi precursors. Field application of naked RNAs is generally impractical due to the sensitivity of RNA to environmental specific and non-specific degradation. Furthermore, RNA is highly susceptible to degradation during the course of feeding and in transit through the insect gut. The highly stable form of VLPs serves to protect RNA borne within the VLPs in vitro. The question remains, are VLPs capable of effectively delivering RNAi precursors to the RNAi processing pathways, such as Dicer, of target insects? In particular, can VLPs protect RNAi precursors within the insect digestive tract and still deliver the intact RNAi precursor to the RNAi processing pathway of the target insect? The results presented here indicate that VLPs are extremely effective at delivering RNAi precursors into target insects.

An important advantage of producing RNAi precursors by the methods described here is that costly and complicated in vitro synthesis of RNA precursors is avoided and the desired RNA constructs can be produced by simple and economic fermentation methods. Production and purification of large quantities of RNAi precursors is facilitated by optionally coupling synthesis of the desired polynucleotide with expression of self-assembling bacteriophage capsid proteins, such as those of bacteriophage Qβ or MS2, to produce easily purified and relatively stable ARCs (VLPs), which may be applied directly to plant surfaces upon which the targeted insect pests feed, for example by spraying.

Once ingested, the ARCs may be digested in the course of transiting the insect host gut and the RNA molecules absorbed by cells lining the gut. Within the target insect cells the RNAi precursors are processed by, among other things, the host Dicer enzyme complex to generate effective RNAi forms targeted against host gene transcripts to suppress expression of essential host genes. Examples of such essential genes include, without limitation, genes involved in controlling molting or other larval development events, actin or other cellular structural components, as well as virtually any gene related to replication, transcription or translation or other fundamental process required for viability.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises DNA sequences, which when transcribed produce RNAi precursor molecules and mRNA translated into bacteriophage coat protein, which together, are incorporated into uniquely stable VLPs. The VLPs may be purified in a form suitable for ingestion by feeding insects. Once ingested by the target insects, the VLPs transit the gut where they are then assimilated into the insect cells where the RNAi precursor is processed into a form of RNAi that suppresses expression of a target gene important to insect viability. In some embodiments, suppression of such target genes is designed to result in death of the target insect. In another embodiment, suppression of target genes is designed to produce sterile off-spring. A key feature of the VLPs is that they are stable enough to protect the encapsidated RNAi precursors from degradation by non-specific environmental agents or by insect target cell RNAse enzymes, but remain capable of introducing the RNAi precursors into the RNAi pathways in target insect cells after they are ingested.

Example sequences presented here are designed to be ligated into suitable bacterial plasmid vectors as AsiSI-NotI digested DNA fragments. Such DNA sequence fragments can be produced by direct synthesis or by sub-cloning the constituent fragments using techniques well known to those skilled in the art. The specific sequences may be modified as desired to manipulate specific restriction enzyme sites, incorporate alternative ribozyme binding sites, accommodate alternative bacteriophage pac sequences, and the specificity of the RNAi sequences may be modified to target different genes and insect hosts. Bacterial plasmid vectors containing transcriptional promoters capable of inducibly transcribing these DNA sequences include, without limitation, bacteriophage T7 gene 1 promoter, bacteriophage T5 promoter, and the bacteriophage lambda PL and PR promoters. Bacterial plasmid vectors may also contain the bacteriophage Qβ or bacteriophage MS2 capsid protein coding sequence expressed from an inducible promoter. Alternatively, such inducibly expressed capsid proteins may be present on a separate bacterial plasmid compatible with the bacterial plasmid carrying the inducible cargo RNA sequences.

The production and purification of VLPs containing RNA cargo molecules and recovery of the RNA cargo molecules are described in detail in U.S. Patent Application Publication Nos. 2013/0208221 (at least paragraphs 0013 and 0014), 2014/0302593 (at least paragraphs 0016, 0052, 0065 and 0085-0086), and as described in U.S. Pat. No. 9,181,531 (passim), the contents of each incorporated herein by reference. In addition, related methods are also described in U.S. Patent Application Publication Nos. 2010/0167981 and 2012/0046340, PCT/US2012/071419 and PCT/US2014/041111, and U.S. Pat. Nos. 5,443,969, and 6,214,982, the contents of each are also incorporated herein by reference. The VLPs produced by these methods can be processed in a number of different ways known to those skilled in the art to facilitate application of such material onto plants and for use in the field. In one embodiment the purified ARCs are further processed for spraying operations. Such processing may include spray drying, introduction of stabilizing or wetting agents, or forming an admixture of VLPs with other desired agents prior to application. Field applications may involve ground or arial spray methods or spot application.

A person skilled in the art will understand that the invention may be targeted to different genes in different insect hosts by modifying the sequences from those described in the Examples to reflect the sequences of the targeted genes in the targeted host organisms. Thus, the invention provides those skilled in the art with a tool for determining the best RNAi target for suppressing a particular gene in any given host cell and a means for producing large quantities of such RNAis. Further, the invention provides for methods of empirically determining which gene or group of genes may constitute the most effective RNAi target within a single insect or group of insects by screening the effectiveness of VLPs containing various RNAi precursors targeted to specific genes or gene combinations in such insects by combinatory cloning methods. The invention also supports methods combining VLPs effective for control of certain insects in the field with different VLPs effective for control of other insects at the point of application, in order to tailor the insect control properties to those relevant at the point of application. The different insects may be of a different order, genus or species as those targeted by the original VLPs, or may comprise RNAi resistant, or combinations of RNAi resistant populations, wherein the combination of one or more VLPs targeting different genes within the target insect population ensures that no combination of RNAi resistance is likely to occur.

In one embodiment of the present invention, a first DNA sequence within a bacterial host is transcribed to produce a first RNA molecule encoding a bacteriophage coat protein, and a second DNA sequence within said bacterial host is transcribed to produce a second RNA molecule comprising a bacteriophage pac site, followed by an antisense sequence of a target gene from an insect, followed by a unique RNA sequence capable of forming a single-stranded loop, followed by a sense sequence complementary to the antisense sequence of the target gene sequence, followed by a second bacteriophage pac site. The first RNA molecule is an mRNA which is translated by the bacterial host to produce a plurality of bacteriophage coat protein which, in combination with the second RNA molecule comprising the bacteriophage pac sequences, spontaneously forms a VLP, wherein the second RNA molecule is packaged within the VLP. VLPs are isolated and purified prior to application to the outer surfaces of a plant. Target insects feeding upon the plant ingest the VLP which in turn introduces the RNA molecule borne within the VLP into the host insect cells where it is processed by the host insect cell's endogenous RNAi pathways, resulting in RNAi-mediated suppression of gene expression of the host insect target gene. In one embodiment the insect is of the order Coleoptera. In preferred embodiments the Coleopteran insect is a Colorado potato beetle.

In another embodiment of the present invention, a first DNA sequence within a bacterial host is transcribed to produce a first RNA molecule encoding a bacteriophage coat protein, and a second DNA sequence within said bacterial host is transcribed to produce a second RNA molecule comprising a bacteriophage pac site, followed by an antisense sequence of a target gene from an insect, optionally followed by one or more bacteriophage pac sites. The first RNA molecule is an mRNA which is translated by the bacterial host to produce a plurality of bacteriophage coat protein which, in combination with the second RNA molecule comprising the bacteriophage pac sequences, spontaneously forms VLPs, wherein the second RNA molecule is packaged within the VLP. The VLPs are isolated and purified prior to application to the outer surfaces of a plant. Target insects feeding upon the plant ingest the VLP which in turn introduces the RNA molecule borne within the VLP into the host insect cells where it results in anti-sense RNA-mediated suppression of gene expression of the host insect target gene. In one embodiment the insect is of the order Coleoptera. In preferred embodiments the Coleopteran insect is a Colorado potato beetle.

In another embodiment, a series of host bacteria containing a first DNA sequence encoding a bacteriophage coat protein and different second DNA sequences encoding various RNAi sequences are isolated. Each isolated host bacteria is clonally expanded and bacterial cell line archived. A sample of each bacterial cell line is subsequently outgrown and induced to transcribe the first and second DNA sequences, the VLPS are allowed to assemble within the host bacteria and the VLPs isolated therefrom. The RNA sequences within the series of resulting VLPs each encode a different antisense and optionally a complementary sense sequence homologous to different insect target genes or on different regions of a given insect target gene or on target genes from different insect targets altogether. Each of the different VLPs produced by the series of host bacteria is fed to target insects and their ability to suppress host insect gene expression is measured, for example by scoring target insect mortality. Those VLPs producing the greatest level of RNAi-mediated suppression of gene expression represent the most effective RNA target for that particular target insect or position within a given target insect gene. Recourse to the corresponding bacterial cell line that produced each VLP allows quick identification of the corresponding target sequence or gene. Likewise, recourse to the corresponding host bacterial cell line facilitates rapid scale-up of the desired VLP for RNAi-mediated suppression of gene expression of the host insect target gene for field application or further experimental investigation. One skilled in the art will recognize that random or pseudo-random collections of complementary DNA sequences based on insect genomic sequence data or for subsets of such genomic sequence encoding likely essential genes can be screened using multiplex or automated cloning technologies.

EXAMPLES

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The publications discussed throughout are provided solely for their disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The following examples are included to demonstrate the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the disclosure. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes could be made in the disclosure and still obtain a like or similar result without departing from the spirit and scope of the disclosure, therefore all matter set forth is to be interpreted as illustrative and not in a limiting sense.

Example 1 Efficacy of Colorado Potato Beetle Control by VLPs Containing an RNAi Precursor

To determine whether VLPs containing a dsRNAi precursor targeting the β-actin gene of Colorado potato beetle (another Coleopteran insect) can suppress β-actin expression as effectively as the naked dsRNAi precursor, the following study was carried out. A 294 bp fragment of beta actin from Colorado potato beetle (Leptinotarsa decemlineata strain Freeville actin mRNA, GenBank sequence ID: gb|KJ577616.1, nucleotides 1-294) was cloned into pAPSE10136 (SEQ ID NO: 1) in such a way as to produce a transcript with sequences comprising both corresponding sense and anti-sense strands separated by a short loop of non-homologous sequence. This RNA represents a 294 bp dsRNAi precursor targeted against beta actin. The dsRNAi precursor DNA sequence was produced by PCR amplification of the 294 bp region of interest from Colorado potato beetle chromosomal DNA using primers 1174 (SEQ ID NO: 2) and 1175 (SEQ ID NO: 3), the PCR product was ligated into an intermediate plasmid by digestion of the PCR fragment and vector with restriction endonucleases AsiSI and PmeI in the sense orientation relative to one of the vector encoded T7 gene 1 promoter. The loop and anti-sense sequences were produced from the sense-strand DNA fragment by PCR amplification with primers 1213 (SEQ ID NO: 4) and 1203 (SEQ ID NO: 5). The resulting PCR fragment and the intermediate plasmid were digested with PmeI and RsrII and ligated together. The desired recombinant plasmids encoding the β-actin sense and antisense strand sequences connected by a short linker expressed from a T7 gene promoter were identified by restriction digest screening. The desired plasmid, pAPSE10216 (SEQ ID NO: 6) was transformed into chemically competent HTE115 (DE3) cells and individual clones selected for ampicillin resistant transformants.

Ampicillin resistant transformants were selected on LB agar plates containing 100 micrograms/ml ampicillin. The selected clones were subsequently grown at 37° C. in 100 ml of LB media containing ampicillin until the culture reached OD600 0.8, at which time isopropyl β-D-thiogalactopyranoside was added to a final concentration of 1 mM to induce T7 polymerase directed transcription of MS2 capsid protein and the 294 bp siRNA precursor. The induced cultures were allowed to grow for at least 4 hours post-induction to allow sufficient time for VLP formation. Cells were collected by centrifugation at 3,000 g at 4 C. Each pellet was stored at 4° C. until processing.

VLPs containing the 294 bp siRNA precursor were purified by re-suspending each pellet in approximately 10 volumes of 20 mM Tris-HCl, pH 7.0, containing 10 mM NaCl and sonicated to lyse the cells. Cell debris was removed by centrifugation at 16,000 g. Each sample was further processed by addition of Benzonase® Nuclease (Sigma Aldrich, St. Louis, Mo.) added to a final concentration of about 100 units per mL and incubated at 37° C. for two hours. Proteinase K was then added to final concentration of 150 micrograms per mL and incubated at 37° C. for an additional three hours. A saturated ammonium sulfate solution was prepared by adding ammonium sulfate to water to a final concentration of 4.1 M. The saturated ammonium sulfate was added to the enzymatically treated VLPs to a final concentration of 186 mM (approximately a 1:22 dilution) and placed on ice for two hours. Unwanted precipitate was cleared from the lysate by centrifugation at 16,000 g. A second precipitation was conducted by addition of 155 mg of dry ammonium sulfate directly to each mL of cleared lysate. Each sample was vortexed and incubated on ice for two hours. Each precipitate was spun down at 16,000 g and the solid precipitate resuspended in one tenth the original volume of 20 mM Tris-HCl, pH 7.0, containing 10 mM NaCl.

The resuspended VLPs were used to test the efficacy of encapsidated RNAi on Colorado potato beetle larvae relative to the corresponding naked RNAi. Each experimental and control cohort included 10 individual beetles undergoing 10 identical treatments. Each treatment or control sample was applied in 50 μl droplets to the surface of a 1 cm diameter potato leaf disc. Each time an application was made, a clean pipette tip was used. The treatment was allowed to dry on the leaf surface prior to being presented to the larvae. During a pretreatment period, all food was removed from the larval containers and larvae were starved for 2 hours before introduction of treated leaves to the larvae. After the starvation period, one larva was placed on each treated potato leaf in a petri dish, where it was allowed to feed on the disc until the leaf tissue was completely devoured. Larvae were allowed to feed at three separate times on treated potato leaves every two days, given a normal diet of potato leaves in the interim and monitored for mortality on a daily basis up to 21 days post-treatment. After the final treatment, live larvae were maintained on untreated potato leaves for an additional 21 days.

Table 1 summarizes the results of treating Colorado potato beetle larvae with the test RNAi administered as naked RNA or encapsidated in an ARC, produced from pAPSE10216. In addition, VLPs containing random E. coli derived RNAs with no significant homology to the Colorado potato beetle beta-actin were included as a control of general non-specific VLP toxicity. These results indicate that these VLP encapsidated RNAs are as effective in killing Colorado potato beetle larvae by suppressing expression of the essential actin gene as unencapsidated RNAi:

TABLE I Summary of mortality rates for Colorado potato beetle (Leptinotarsa decemlineata) larvae treated with RNA and VLP formulations Maximum Days to reach Dose mortality maximum Treatment (microgram) (%) mortality Untreated control 0 20 15 Water control 0 20 16 VLPs with 0.5 30 19 unrelated dsRNA VLPs with dsRNAi 0.5 100 1 precursor dsRNAi precursor 0.5 100 1 without VLP

The naked dsRNA treated controls exhibit a high degree of mortality, consistent with the hypothesis that suppression of actin gene expression by this dsRNA results in death of beetle larvae that consume it. The cohort treated with VLPs containing the unrelated RNA exhibit little or no mortality, indicating that VLPs are not inherently toxic to the beetle larvae. The ARCs provide an effective delivery platform for RNAi active molecules, and the high level of mortality verifies that the packaging and processing steps for manufacturing VLPs does not inhibit effectiveness of the RNAi response observed from such dsRNA.

Additional experiments at doses lower than 0.5 μg, e.g. at 0.05 μg, reveal that ARCs with actin hairpin RNA have similar efficacy at lower doses to naked dsRNA at higher doses targeting the same actin sequence.

The ability of these constructs to kill Colorado potato beetle larvae confirms that these ARCs are an effective tool for introducing targeted RNAi precursors into an insect host and that these precursors can be properly processed by the host cell RNAi pathway to suppress gene expression of the target gene. These results directly demonstrate that ARCs comprising siRNA precursors are an effective delivery system for controlling Colorado potato beetle and Coleopteran insects generally.

Example 2 Efficacy of Controlling Colorado Potato Beetle Larvae by VLPs Containing Single Stranded Antisense RNA

To test whether anti-sense RNA (ssRNAi) can be effectively delivered to target insects by use of VLPs, a 294 bp DNA sequence fragment corresponding to a portion of the beta actin gene of Colorado potato beetle (Leptinotarsa decemlineata strain Freeville actin mRNA, GenBank sequence ID: gb|KJ577616.1, nucleotides 1-294) was constructed from primers 1176 (SEQ ID NO: 7) and 1177 (SEQ ID NO: 8). The primers were ordered from IDT (Integrated DNA Technologies, Inc., Coralville, Iowa) and used to amplify the beta actin sequence fragment from Colorado potato beetle genomic DNA by Accuprime PCR while adding an AsiSI restriction site 5′ of the beta actin sequence fragment and a PmeI restriction site 3′ of the beta actin sequence fragment. The resulting PCR product was digested with AsiSI and PmeI restriction endonucleases and subsequently ligated into pAPSE10136 (SEQ ID NO: 1) previously treated with AsiSI and PmeI, in the anti-sense orientation relative to the upstream T7 promoter, to form pAPSE10190 (SEQ ID NO: 9). This plasmid allows the β-actin antisense strand RNA (ssRNAi) to be packaged in VLPs at high efficiency by incorporating bacteriophage pac sites into the transcript. Chemically competent HTE115 (DE3) cells were transformed and VLPs were produced by fermentation and subsequently isolated as described in Example 2. The VLPs were then tested for the ability to suppress Colorado potato beetle larvae as described in Example 2. Table 2 summarizes the results:

TABLE 2 Summary of mortality rates of Colorado potato beetle (Leptinotarsa decemlineata) larvae treated with single stranded anti-sense RNA and VLP formulations. Maximum Days to reach Dose mortality maximum Treatment (microgram) (%) mortality Untreated control 0 20 15 Water control 0 20 16 VLPs with no ssRNAi 0.5 30 19 VLPs with ssRNAi 0.5 100 1 ssRNAi without VLP 0.5 100 8

These data indicate that VLPs improve the efficacy of single-stranded anti-sense RNA directed to suppressing expression of the essential beta-actin gene in killing Colorado potato beetle larvae. Further, these results indicate that these VLPs are even more effective in killing Colorado potato beetle larvae by suppressing expression of the essential actin gene than the corresponding unencapsidated RNAi. These results suggest that ARCs comprising antisense ssRNA also serve as an effective delivery system for controlling Coleopteran insects generally, and Colorado potato beetle specifically.

SEQUENCES SEQ ID NO: Sequence Description 1 ttctcatgtt tgacagctta Plasmid tcatcgataa gctttaatgc pAPSE ggtagtttat cacagttaaa 10136 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatgaa ttcagatctc gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc ctctagatca caagtttgta caaaaaagca ggctaagaag gagatataca tacgccggcc attcaaacat gaggattacc catgtattta aatacccatg tccaggcgcg ctccgcgatc gcacgcggac aactactaca gggtttaaac ctttcggatt ataacatcac atctaggcgc gcctgacgat caaccatacc agacggaccg aatacccggt ctgaacgagg gcggccgcgg tacccaagaa gtacttagag ttaattaagg agttcaaaca tgaggatcac ccatgtcgaa gctcccacac cctagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg tggtcgccat gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc ggcgggcatg catcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg cgaccacacc cgtcctgtgg atccagatct cgatcccgcg aaattaatac gactcactat agggagacca caacggtttc cctctagatc acaagtttgt acaaaaaagc aggctaagaa ggagatatac atatggcgtc taactttacc caattcgttc tggttgataa cggcggtacg ggtgacgtta ccgtagctcc gtccaacttc gccaacggtg ttgcggaatg gattagctct aacagccgct ctcaggccta caaagtcacg tgctccgttc gtcagtctag cgcgcagaat cgcaaataca ccatcaaagt tgaagtaccg aaagtcgcaa cgcagaccgt aggcggcgta gaactcccag ttgcggcctg gcgctcttac ctcaacatgg aactgactat tccgattttt gcgacgaact ccgactgcga actgattgtt aaggcaatgc agggcctgct gaaagacggt aatccgatcc catctgcaat cgctgctaac tctggcattt actaataagc ggacgcgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta tgattcttct cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa cttcgatcat tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga acgggttggc atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttgg cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaa 2 cattggcgat cgcgcacgag PCR primer gtttttctgt ctagtgagca 1174 g 3 cattggttta aactcatccc PCR primer agttggtgat gataccg 1175 4 cattggttta aaccctctag CR primer ctgctttaca aagtactggt 1213 tccctttcca gcgggatgct ttatctaaac gcaatgagag aggtattcct caggccacat cgcttcctag ttccgctggg atccatcgtt ggcggccgaa gccgccattc catagtgagt tctggcgcgc ctcatcccag ttggtgatga taccgtgttc 5 cattgcggtc cggcacgagg PCR primer tttttctgtc tagtgag 1203 6 ttctcatgtt tgacagctta plasmid tcatcgataa gctttaatgc pAPSE ggtagtttat cacagttaaa 10216 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatgaa ttcagatctc gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc ctctagatca caagtttgta caaaaaagca ggctaagaag gagatataca tacgccggcc attcaaacat gaggattacc catgtattta aatacccatg tccaggcgcg ctccgcgatc gcgcacgagg tttttctgtc tagtgagcag tgtccaacct caaaagacaa catgtgtgac gacgatgtag cggctcttgt cgtagacaat ggatccggta tgtgcaaagc cggtttcgca ggagatgacg caccccgtgc cgtcttcccc tcgatcgtcg gtcgcccaag gcatcaagga gtcatggtcg gtatgggaca aaaggactca tacgtaggag atgaagccca aagcaaaaga ggtatcctca ccctgaaata ccccatcgaa cacggtatca tcaccaactg ggatgagttt aaaccctcta gctgctttac aaagtactgg ttccctttcc agcgggatgc tttatctaaa cgcaatgaga gaggtattcc tcaggccaca tcgcttccta gttccgctgg gatccatcgt tggcggccga agccgccatt ccatagtgag ttctggcgcg cctcatccca gttggtgatg ataccgtgtt cgatggggta tttcagggtg aggatacctc ttttgctttg ggcttcatct cctacgtatg agtccttttg tcccataccg accatgactc cttgatgcct tgggcgaccg acgatcgagg ggaagacggc acggggtgcg tcatctcctg cgaaaccggc tttgcacata ccggatccat tgtctacgac aagagccgct acatcgtcgt cacacatgtt gtcttttgag gttggacact gctcactaga cagaaaaacc tcgtgccgga ccgaataccc ggtctgaacg agggcggccg cggtacccaa gaagtactta gagttaatta aggagttcaa acatgaggat cacccatgtc gaagctccca caccctagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg ggcggcgggc atgcatcgtc cattccgaca gcatcgccag tcactatggc gtgctgctag cgctatatgc gttgatgcaa tttctatgcg cacccgttct cggagcactg tccgaccgct ttggccgccg cccagtcctg ctcgcttcgc tacttggagc cactatcgac tacgcgatca tggcgaccac acccgtcctg tggatccaga tctcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag atcacaagtt tgtacaaaaa agcaggctaa gaaggagata tacatatggc gtctaacttt acccaattcg ttctggttga taacggcggt acgggtgacg ttaccgtagc tccgtccaac ttcgccaacg gtgttgcgga atggattagc tctaacagcc gctctcaggc ctacaaagtc acgtgctccg ttcgtcagtc tagcgcgcag aatcgcaaat acaccatcaa agttgaagta ccgaaagtcg caacgcagac cgtaggcggc gtagaactcc cagttgcggc ctggcgctct tacctcaaca tggaactgac tattccgatt tttgcgacga actccgactg cgaactgatt gttaaggcaa tgcagggcct gctgaaagac ggtaatccga tcccatctgc aatcgctgct aactctggca tttactaata agcggacgcg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca ggcaggtaga tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc taacttcgat cattggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat ggaacgggtt ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa 7 cattggcgat cgctcatccc PCR primer agttggtgat gataccg 1176 8 cattggttta aacgcacgag PCR primer gtttttctgt ctagtgag 1177 9 ttctcatgtt tgacagctta plasmid tcatcgataa gctttaatgc pAPSE ggtagtttat cacagttaaa 10190 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatgaa ttcagatctc gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc ctctagatca caagtttgta caaaaaagca ggctaagaag gagatataca tacgccggcc attcaaacat gaggattacc catgtattta aatacccatg tccaggcgcg ctccgcgatc gctcatccca gttggtgatg ataccgtgtt cgatggggta tttcagggtg aggatacctc ttttgctttg ggcttcatct cctacgtatg agtccttttg tcccataccg accatgactc cttgatgcct tgggcgaccg acgatcgagg ggaagacggc acggggtgcg tcatctcctg cgaaaccggc tttgcacata ccggatccat tgtctacgac aagagccgct acatcgtcgt cacacatgtt gtcttttgag gttggacact gctcactaga cagaaaaacc tcgtgcgttt aaacctttcg gattataaca tcacatctag gcgcgcctga cgatcaacca taccagacgg accgaatacc cggtctgaac gagggcggcc gcggtaccca agaagtactt agagttaatt aaggagttca aacatgagga tcacccatgt cgaagctccc acaccctagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc gagcaggact gggcggcggg catgcatcgt ccattccgac agcatcgcca gtcactatgg cgtgctgcta gcgctatatg cgttgatgca atttctatgc gcacccgttc tcggagcact gtccgaccgc tttggccgcc gcccagtcct gctcgcttcg ctacttggag ccactatcga ctacgcgatc atggcgacca cacccgtcct gtggatccag atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta gatcacaagt ttgtacaaaa aagcaggcta agaaggagat atacatatgg cgtctaactt tacccaattc gttctggttg ataacggcgg tacgggtgac gttaccgtag ctccgtccaa cttcgccaac ggtgttgcgg aatggattag ctctaacagc cgctctcagg cctacaaagt cacgtgctcc gttcgtcagt ctagcgcgca gaatcgcaaa tacaccatca aagttgaagt accgaaagtc gcaacgcaga ccgtaggcgg cgtagaactc ccagttgcgg cctggcgctc ttacctcaac atggaactga ctattccgat ttttgcgacg aactccgact gcgaactgat tgttaaggca atgcagggcc tgctgaaaga cggtaatccg atcccatctg caatcgctgc taactctggc atttactaat aagcggacgc gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga a 

What is claimed is:
 1. A virus-like-particle (VLP) for controlling target insects, the VLP comprising a bacteriophage capsid protein encapsidating an RNA transcript comprising at least one bacteriophage pac sequence coupled to an RNAi precursor sequence targeted against a gene transcript of the insect to suppress expression of the gene in the insect.
 2. The VLP of claim 1, wherein the bacteriophage capsid protein derives from a levivirus.
 3. The VLP of claim 2, wherein the levivirus is Qβ.
 4. The VLP of claim 2, wherein the levivirus is MS2.
 5. The VLP of claim 1, wherein the RNAi precursor forms an siRNA.
 6. The VLP of claim 1, wherein the RNAi precursor forms an antisense RNA.
 7. The VLP of claim 1, wherein the RNA transcript comprises a first bacteriophage pac site, followed by an antisense sequence of the gene, followed by an RNA sequence capable of forming a single-stranded loop, followed by a sense sequence complementary to the antisense sequence of the gene, followed by a second bacteriophage pac site.
 8. The VLP of claim 1, wherein the RNA transcript comprises a bacteriophage pac site, followed by an antisense sequence of the gene, optionally followed by one or more bacteriophage pac sites.
 9. The VLP of claim 1, wherein the gene is an essential gene.
 10. The VLP of claim 9, wherein the essential gene is a gene controlling a larval development event, a gene encoding cellular structural components, or a gene encoding nucleic acid replication, transcription, or translation.
 11. The VLP of claim 9, wherein suppression of the essential gene results in death of the insect.
 12. The VLP of claim 9, wherein suppression of the essential gene results in the production of sterile off-spring.
 13. The VLP of claim 9, wherein the essential gene encodes β-actin.
 14. The VLP of claim 1, wherein the target insect is a Lepidopteran insect.
 15. The VLP of claim 1, wherein the target insect is a Coleopteran insect.
 16. The VLP of claim 15, wherein the Coleopteran insect is bark beetle, elm leaf beetle, Asian longhorn beetle, death watch beetle, mountain pine beetle, coconut hispine beetle, a corn rootworm, or Colorado potato beetle.
 17. The VLP of claim 1, wherein the target insect is Colorado potato beetle.
 18. The VLP of claim 17, wherein the gene encodes β-actin, and the RNAi precursor sequence is encoded by nucleotides 314-1219 of SEQ ID NO:
 6. 19. The VLP of claim 17, wherein the gene encodes β-actin, and the RNAi precursor sequence is encoded by nucleotides 317-820 of SEQ ID NO:
 9. 20. A combination of two or more VLPs of claim 1, wherein each RNAi precursor sequence is targeted against a different gene transcript of the insect to suppress expression of each targeted gene in the insect. 